- Original article
- Open Access
Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining
- Minjie Zhang†1, 2,
- Liu Wang†3,
- Ke An†1, 2,
- Jun Cai1,
- Guochao Li1, 2,
- Caiyun Yang1,
- Huixian Liu1,
- Fengxia Du1,
- Xiao Han1, 2,
- Zilong Zhang1, 2,
- Zitong Zhao1, 2,
- Duanqing Pei4,
- Yuan Long5,
- Xin Xie5,
- Qi Zhou3 and
- Yingli Sun1Email authorView ORCID ID profile
- Received: 31 October 2017
- Accepted: 11 June 2018
- Published: 25 July 2018
Abstract
Background
Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) share many common features, including similar morphology, gene expression and in vitro differentiation profiles. However, genomic stability is much lower in iPSCs than in ESCs. In the current study, we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis.
Methods
Mouse iPSCs, ESCs and embryonic fibroblasts were exposed to ionizing radiation (4 Gy) to introduce double-strand DNA breaks. At 4 h later, fidelity of DNA damage repair was assessed using whole-genome re-sequencing. We also analyzed genomic stability in mice derived from iPSCs versus ESCs.
Results
In comparison to ESCs and embryonic fibroblasts, iPSCs had lower DNA damage repair capacity, more somatic mutations and short indels after irradiation. iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair. Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice.
Conclusions
The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due, at least in part, to low fidelity of DNA damage repair.
Keywords
- Genomic stability
- DNA damage repair
- iPSCs
- ESCs
Background
Embryonic stem cells (ESCs) are pluripotent and could differentiate into all types of somatic cells [1]. ESCc have enormous potential in the treatment of a variety of diseases, but their clinical application has been limited by ethical controversy. In 2006, Yamanaka and colleagues overexpressed four transcription factors (Oct4, Sox2, c-Myc and Klf4) in mouse somatic cells and obtained ESC-like pluripotent stem cells, termed induced pluripotent stem cells (iPSCs) [2]. iPSCs resemble ESCs in morphology, gene expression profile, epigenetic status and in vitro differentiation capacity. The development of iPSCs raises new hope for personalized clinical therapy [3–5].
The four transcription factors (Oct4, Sox2, c-Myc and Klf4) that are critical for the production of iPSCs are frequently overexpressed in various cancers, and mice derived from iPSCs are prone to develop tumors [6–9]. Although only a small population of transformed cells with genetic mutations is likely to develop into tumors [10], the genomic instability of iPSCs is a major concern that could produce huge impact on their eventual clinical use [11–16].
One possible explanation for the observed greater genomic instability of iPSCs is alterations in the fidelity of DNA repair pathways. Double-stranded DNA breaks, for example, can be repaired via homologous recombination (HR) with high fidelity, or via non-homologous end joining (NHEJ) with lower fidelity [17–20]. In the current study, we examined whether iPSCs differ from other types of pluripotent cells in their ability to perform these types of DNA repair. Briefly, ionizing radiation was used to induce double-stranded DNA breaks in the following cells: mouse iPSCs induced using lentivirus (lv-iPSCs) or chemically with CHR99021, Repsox and forskolin (ci-iPSCs) [21]; mouse ESCs; and mouse embryonic fibroblasts (MEFs) [22–26].
The experiments showed that lv-iPSCs are more likely than the other cell types to harbor genomic abnormalities, likely due to lower genomic fidelity of DNA damage repair. We also found greater genomic stability in ci-iPSCs than lv-iPSCs.
Methods
Cell lines and culture
The lv- and ci-iPSCs were derived from female transgenic OG2 mice carrying an Oct4-GFP transgene. Both types of iPSCs and ESCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 15% fetal bovine serum (FBS; Gibco), 1% MEM non-essential amino acids (Gibco), 1% penicillin/streptomycin (Gibco), 2 mmol/L l-glutamine (Gibco), 1 × 103 units/mL of mouse leukemia inhibitory factor (Millipore, Temecula, CA, USA) and 0.1 mmol/L 2-mercaptoethanol (Gibco) [27]. The medium was changed daily, and cells were passaged every 2 days using 0.25% trypsin (Thermo Fisher Scientific, Beijing, China) [28]. MEFs were cultured in DMEM supplemented with 15% FBS, 1% non-essential amino acids and 1% penicillin/streptomycin [29].
Irradiation
Cells were passaged 1 day before γ-irradiation (4 Gy) with a cobalt irradiator (Thermo Fisher Scientific). After the irradiation, cells were immediately returned to the incubator, and cultured for 4 h prior to analyses as described below.
Western blotting
To test the phosphorylation level of ATM, cells were lysed in ATM lysis buffer [20 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 0.2% Tween-20, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 2 mmol/L dithiothreitol, 50 mmol/L NaF, 500 μmol/L NaVO4, 1 mmol/L phenylmethylsulfonyl fluoride, 0.1 μg/mL aprotinin and 0.1 µg/mL leupeptin], and centrifuged, as describe previously [30].
In assays of histone modification, cells were re-suspended in 1-mL triton extraction buffer (TEB) containing 0.5% Triton X-100 and 2 mmol/L PMSF, and then lysed on ice for 10 min. The lysates were centrifuged at 1500g for 10 min at 4 °C. The pellet was washed with 1.5-mL TEB, re-suspended in 0.2 mol/L HCl, and incubated at 4 °C overnight. Samples were centrifuged at 6500g for 10 min, after which 200-µL supernatant was transferred to a new tube, and neutralized with 20-µL 2 mol/L NaOH.
Samples were separated using SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Blots were incubated with a primary antibody against one of the following proteins: phospho-ATM (1:1000; R&D Systems, Minneapolis, MN, USA), β-actin (1:3000; Beyotime Biotech, Beijing, China), H3 (1:30,000; Abcam, Cambridge, MA, USA) and H3K9me3 (1:3000; Abcam). Blots were washed three times with phosphate-buffered saline (PBS), and then incubated with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:3000; Gene Tex, San Diego, CA, USA) or anti-rabbit secondary antibody (1:3000; Abcam). Protein bands of interest were visualized using an Image Quant ECL system (GE Healthcare, Piscataway, NJ, USA).
Immunofluorescence labeling of γ-H2AX foci
Cells were passaged onto slides, exposed 24 h later to 4 Gy of γ-irradiation, and incubated at 37 °C for 4 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, washed again with PBS, permeabilized for 10 min using 0.05% Triton X-100 and 0.5% NP-40, and then washed three times (5 min each) in PBS. The cells were blocked for 1 h with 2% bovine serum albumin (BSA), and then incubated for 1 h at room temperature with a mouse anti-γH2AX antibody (1:500; Millipore, Temecula, CA, USA). Cells were washed three times with PBS containing 0.05% Tween 20, and then incubated with a goat anti-mouse secondary antibody (1:800; Abcam) for 1 h in the dark at room temperature. Cells were counterstained with 0.2 mg/mL 4′,6-diamidino-2-phenylindole (DAPI, 1:2000; Sigma, Shanghai, China). Confocal images were acquired and analyzed using a TCS SP5 (Leica) microscope equipped with an HCX PL 63 × 1.4 CS oil-immersion objective lens.
DNA extraction
Three types of cells (lv-iPSCs, ci-iPSCs, ESCs) were digested with 0.25% trypsin and re-suspended in gelatin-coated dishes. After incubation at 37 °C for 15 min, supernatants were transferred to 15-mL centrifuge tubes, and cells were collected by centrifugation at 500g for 5 min at room temperature. DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
Whole-genome re-sequencing
Whole-genome DNA libraries suitable for sequencing using an Illumina sequencing platform were generated from 1-µg genomic DNA. The DNA was sheared to approximately 300–500 bp using a Covaris S220 instrument (Life Technologies, Carlsbad, CA, USA). A total of 2× 101-bp paired-end reads were produced using the HiSeq 2000 DNA Sequencer.
The sequencing data were mapped to a reference mouse genomic sequence (mm9) using the Burrows–Wheeler alignment tool algorithm [31]. Unique alignment reads were retained for later analysis. Using the untreated cells as a control, single-nucleotide variations (SNVs) were collected using the “mpileup” tool in SAMTools as well as the UnifiedGenotyper in the GATK module [32, 33]. Quality recalibration and local realignment were performed using GATK tools before variation calling was performed. The following criteria were applied for calling mutations using pairwise samples: (1) the minimum coverage of variant sites had to be greater than 20 and base quality greater than 15; (2) the frequency of mutant SNVs had to be 0 in control samples and 0.2 in irradiated samples; and (3) the variant sites had to be supported by at least two reads on the forward strand and two reads on the reverse strand.
RNA sequencing
Total RNA was extracted from each cell line using TRIzol reagent and enriched for mRNA using oligo (dT) magnetic beads. Approximately 1-µg mRNA was fragmented and electrophoresed to isolate mRNA fragments (200–250 bases). These fragments were subjected to end repair, 3′ terminal adenylation and adapter ligation, followed by cDNA synthesis. The resulting cDNAs were gel-electrophoresed to isolate 250–300 bp fragments, and were sequenced using a HiSeq 2000 system (Illumina).
Sequencing reads were aligned to a reference sequence (GRCm37/mm9) using TopHat alignment software [34, 35]. Only uniquely aligned reads were used for transcript assembly, which was performed using Cufflinks software [36]. Read counts for each gene were calculated, and the expression levels of each gene were normalized using the “fragments per kilobase of exon model per million mapped” (FPKM) algorithm. Differentially expressed genes were filtered based on false discovery rate (FDR)-adjusted P < 0.05. The profile of differentially expressed genes was visualized and analyzed using the Bioconductor function “CummeRbund” in the R program [37]. Hierarchical clustering was performed using the “heatmap” package in R.
Generation of iPSC- and ESC-derived mice
Two cell-stage ICR embryos were electrofused to produce tetraploid embryos, and 10–15 iPSCs and ESCs were subsequently injected into the reconstructed tetraploid blastocysts. Embryos were cultured for 1 day prior to transplantation into the uterus of pseudo-pregnant mice. Caesarean sections were performed at E19.5, and the pups were fostered by lactating ICR mothers [38].
Comet assay
Mice derived from iPSCs or ESCs as well as C57 mice were treated with 4 Gy ionizing radiation. At 4 h later, bone marrow cells were isolated and re-suspended using PBS and concentrated by adding 150-μL molten 0.75% low-melting-point agarose. An aliquot of concentrated cells (60 μL) was then added to molten 0.8% normal-melting-point agarose on comet slides. The slides were incubated for 1–2 h at 4 °C with pre-chilled lysis buffer, stored in the dark at 4 °C for 20 min, then incubated with pre-chilled electrophoresis buffer (0.3 mol/L NaOH containing 0.5 mol/L EDTA, pH > 13.0). Gel electrophoresis was performed at 25 V for 20 min at 4 °C. Slides were incubated at 4 °C for 15 min with neutralization buffer (0.4 mol/L Tris, pH > 7.5), washed with 100% ethanol for 3–5 min and air-dried at room temperature. Diluted ethidium bromide (EB) solution (20–30 μL) was placed onto each dried agarose circle. Slides were then read at 100 cells/sample using a fluorescence microscope equipped with CASP DNA damage analysis software.
Results
Similar gene expression profile between lv-iPSCs and ESCs
Gene expression profile of ESCs, lv-iPSCs and MEFs. a Scatter plots used to identify global trends in gene expression and differences among cell lines. b Heat maps showing the expression level of DNA damage repair-associated genes in the cell lines. Blue color indicates lowest expression; fuchsia, highest. c Re-analysis of the expression of DNA damage repair-associated genes during early reprogramming
More DNA mutations in lv-iPSCs than in other cell types after ionizing irradiation
Genomic variation in each cell line after ionizing irradiation. a Circos plot showing genetic alterations in lv-iPSCs, ESCs and MEFs after irradiation, based on the corresponding untreated cells as the reference. CHR, chromosome. b, c Histograms showing the numbers of (b) single-nucleotide variations (SNVs) and (c) short insertions or deletions (indels) in each type of genomic region in each cell line. d Histogram of the number of SNVs in a coding region (CDS) in each cell line
Summary of sequencing results
Parameter | lv-iPSCs | ESCs | MEFs | |||
|---|---|---|---|---|---|---|
IR− | IR+ | IR− | IR+ | IR− | IR+ | |
Total nucleotides sequenced (Gb) | 64.1 | 71.5 | 72.0 | 70.9 | 64.7 | 70.1 |
Genome coverage (fold) | 20× | 21× | 22× | 23× | 20× | 21× |
Total number of reads | 634,852,868 | 708,065,514 | 765,056,092 | 754,255,820 | 640,447,936 | 693,376,402 |
Summary of somatic indels in each cell line
lv-iPSCs | ESCs | MEFs | |
|---|---|---|---|
Somatic indels | 10,127 | 1041 | 679 |
CDS | 7 | 0 | 3 |
Intergenic | 5616 | 594 | 387 |
Intron | 3725 | 370 | 242 |
5′ UTR | 596 | 59 | 31 |
3′ UTR | 180 | 18 | 16 |
Splice site | 3 | 0 | 0 |
Summary of somatic mutations in each cell line
lv-iPSCs | ESCs | MEFs | |
|---|---|---|---|
Somatic mutation | 92,027 | 789 | 2403 |
CDS | 867 | 15 | 24 |
Intergenic | 54,086 | 570 | 1526 |
Intron | 35,567 | 173 | 694 |
5′ UTR | 5128 | 22 | 106 |
3′ UTR | 1379 | 9 | 53 |
Splice site | 0 | 0 | 0 |
CDS | 867 | 15 | 24 |
Synonymous | 559 | 8 | 11 |
Nonsynonymous | 307 | 7 | 13 |
Nonsense | 1 | 0 | 0 |
Frequencies of coding SNVs in ESCs exposed to ionizing radiation
# | Locus | Gene | Mutation | Amino acid change | Freq. IR− (%) | Freq. IR+ (%) |
|---|---|---|---|---|---|---|
1 | Chr1:46244148 | Dnahc7b | T−> C | V−>A | 0.0 | 28.57 |
2 | Chr1:172987924 | Fcgr3 | A−>C | S−>A | 0.0 | 30 |
3 | Chr2:180330052 | Ogfr | A−>G | E−>G | 0.0 | 22.06 |
4 | Chr8:22465823 | Defa-rs1 | C−>G | R−>P | 0.0 | 20 |
5 | Chr10:80640826 | Eef2 | G−>A | E−>K | 0.0 | 30.11 |
6 | Chr15:77465439 | Apol11b | C−>A | K−>N | 0.0 | 20 |
7 | chrX:131293286 | Armcx3 | G−>A | C−>Y | 0.0 | 21.62 |
Frequencies of coding SNVs in MEFs exposed to ionizing radiation
# | Locus | Gene | Mutation | Amino acid change | Freq. IR− (%) | Freq. IR+ (%) |
|---|---|---|---|---|---|---|
1 | Chr1:174406441 | Slamf9 | T−>C | M−>T | 0.0 | 23.68 |
2 | Chr2:10014034 | Kin | G−>A | E−>K | 0.0 | 20.00 |
3 | Chr4:120619977 | Zfp69 | A−>G | S−>P | 0.0 | 20.00 |
4 | Chr4:146162835 | Zfp600 | T−>A | I−>K | 0.0 | 20.00 |
5 | Chr5:72655729 | Atp10d | C−>G | P−>R | 0.0 | 27.78 |
6 | Chr7:25371714 | Zfp575 | G−>A | A−>V | 0.0 | 25.00 |
7 | Chr7:31658366 | Cd22 | C−>T | R−>Q | 0.0 | 30.56 |
8 | Chr7:48249435 | 4930433I11Rik | A−>T | D−>V | 0.0 | 23.08 |
9 | Chr7:48249440 | 4930433I11Rik | G−>C | A−>P | 0.0 | 24.00 |
10 | Chr8:93611040 | Rbl2 | G−>C | R−>T | 0.0 | 20.83 |
11 | Chr14:52076074 | Vmn2r89 | C−>T | A−>V | 0.0 | 20.00 |
12 | Chr18:67019622 | Mc4r | C−>T | G−>S | 0.0 | 22.50 |
13 | Chr18:70668975 | Poli | C−>T | G−>R | 0.0 | 29.03 |
Frequencies of coding SNVs in lv-iPSCs exposed to ionizing radiation
# | Locus | Gene | Mutation | Amino acid change | Freq. IR− (%) | Freq. IR+ (%) |
|---|---|---|---|---|---|---|
1 | chr1:30861639 | Phf3 | C−>G | E−>Q | 0.00 | 20.93 |
2 | chr1:60166069 | Carf | C−>T | R−>W | 0.00 | 42.86 |
3 | chr1:92665043 | Col6a3 | C−>G | E−>D | 0.00 | 22.73 |
4 | chr1:108649819 | Kdsr | G−>T | D−>E | 0.00 | 27.59 |
5 | chr1:152550404 | Hmcn1 | C−>T | V−>I | 0.00 | 25.00 |
6 | chr1:166275528 | Nme7 | G−>A | G−>S | 0.00 | 32.26 |
7 | chr1:171863885 | 1700084C01Rik | G−>A | G−>S | 0.00 | 25.00 |
8 | chr1:175866740 | Ifi203 | G−>A | T−>M | 0.00 | 28.57 |
9 | chr1:186630980 | Mosc1 | G−>C | D−>E | 0.00 | 23.08 |
10 | chr1:186740013 | Mark1 | T−>A | E−>D | 0.00 | 32.14 |
11 | chr2:10112008 | Itih5 | T−>C | S−>P | 0.00 | 42.31 |
12 | chr2:31655794 | Abl1 | A−>G | S−>G | 0.00 | 25.00 |
13 | chr2:31656413 | Abl1 | A−>C | N−>T | 0.00 | 24.24 |
14 | chr2:34634942 | Rabepk | T−>C | K−>E | 0.00 | 44.44 |
15 | chr2:34858984 | Hc | C−>T | S−>N | 0.00 | 20.51 |
16 | chr2:79182476 | Cerkl | C−>T | A−>T | 0.00 | 24.00 |
17 | chr2:86000090 | Olfr1042 | T−>C | T−>A | 0.00 | 42.86 |
18 | chr2:86154881 | Olfr1053 | A−>C | I−>M | 0.00 | 42.86 |
19 | chr2:86828637 | Olfr1101 | T−>G | Q−>P | 0.00 | 41.67 |
20 | chr2:87149857 | Olfr1118 | A−>G | K−>E | 0.00 | 40.74 |
21 | chr2:89033480 | Olfr1226 | G−>A | S−>F | 0.00 | 57.14 |
22 | chr2:90749357 | Kbtbd4 | A−>G | I−>V | 0.00 | 36.36 |
23 | chr2:90894429 | Psmc3 | A−>G | T−>A | 0.00 | 23.53 |
24 | chr2:91757766 | Ambra1 | G−>A | R−>Q | 0.00 | 60.71 |
25 | chr2:92815310 | Prdm11 | G−>A | S−>L | 0.00 | 43.33 |
26 | chr2:119346136 | Exd1 | T−>A | H−>L | 0.00 | 35.00 |
27 | chr2:119577973 | Ltk | C−>T | G−>E | 0.00 | 34.38 |
28 | chr2:120104674 | Pla2g4d | G−>A | P−>L | 0.00 | 20.00 |
29 | chr2:120265164 | Ganc | C−>G | I−>M | 0.00 | 40.00 |
30 | chr2:120357660 | Zfp106 | G−>T | Q−>K | 0.00 | 42.31 |
31 | chr2:126412071 | Slc27a2 | G−>T | A−>S | 0.00 | 24.00 |
32 | chr2:127182455 | Astl | C−>T | P−>L | 0.00 | 30.56 |
33 | chr2:127267842 | Fahd2a | C−>A | G−>W | 0.00 | 21.74 |
34 | chr2:146172498 | Ralgapa2 | C−>T | V−>I | 0.00 | 25.00 |
35 | chr2:150299134 | Zfp345 | A−>T | L−>Q | 0.00 | 24.00 |
36 | chr2:153757199 | Bpifb3 | G−>A | M−>I | 0.00 | 33.33 |
37 | chr2:157822874 | Tti1 | T−>C | K−>R | 0.00 | 20.59 |
38 | chr2:157832871 | Tti1 | C−>T | S−>N | 0.00 | 21.74 |
39 | chr2:165177990 | Zfp663 | C−>T | R−>Q | 0.00 | 21.74 |
40 | chr2:165880571 | Ncoa3 | G−>A | S−>N | 0.00 | 43.48 |
41 | chr2:174471852 | Zfp831 | T−>C | S−>P | 0.00 | 25.93 |
42 | chr3:19570978 | Trim55 | G−>A | G−>S | 0.00 | 29.17 |
43 | chr3:20127155 | Cpa3 | T−>A | K−>I | 0.00 | 36.36 |
44 | chr3:65861245 | Veph1 | A−>G | S−>P | 0.00 | 25.00 |
45 | chr3:88240586 | Sema4a | G−>A | A−>V | 0.00 | 29.41 |
46 | chr3:94167523 | C2cd4d | G−>C | R−>P | 0.00 | 22.22 |
47 | chr3:96096266 | Fcgr1 | G−>A | P−>S | 0.00 | 20.00 |
48 | chr3:97414088 | Chd1l | T−>C | S−>G | 0.00 | 42.86 |
49 | chr3:105789443 | Ovgp1 | C−>T | T−>I | 0.00 | 21.74 |
50 | chr3:116192199 | Rtcd1 | C−>T | V−>I | 0.00 | 27.27 |
51 | chr3:118377426 | Dpyd | G−>A | S−>N | 0.00 | 41.67 |
52 | chr3:137770265 | Mttp | T−>A | T−>S | 0.00 | 26.09 |
53 | chr3:142271248 | Gbp1 | G−>A | E−>K | 0.00 | 30.95 |
54 | chr4:57660898 | Palm2 | G−>A | V−>I | 0.00 | 40.00 |
55 | chr4:106415886 | Fam151a | G−>A | R−>Q | 0.00 | 51.85 |
56 | chr4:116265516 | Gpbp1l1 | T−>A | S−>T | 0.00 | 25.00 |
57 | chr4:118154980 | Tie1 | T−>C | D−>G | 0.00 | 30.30 |
58 | chr4:119804939 | Hivep3 | T−>C | L−>P | 0.00 | 25.00 |
59 | chr4:120620061 | Zfp69 | T−>C | T−>A | 0.00 | 40.00 |
60 | chr4:120620067 | Zfp69 | T−>G | T−>P | 0.00 | 37.04 |
61 | chr4:136193988 | Lactbl1 | A−>G | S−>G | 0.00 | 33.33 |
62 | chr4:141674086 | Kazn | C−>T | A−>T | 0.00 | 20.00 |
63 | chr4:147839151 | Mtor | C−>T | R−>C | 0.00 | 25.71 |
64 | chr5:23825901 | Kcnh2 | T−>G | T−>P | 0.00 | 34.78 |
65 | chr5:23905831 | Abcb8 | T−>C | W−>R | 0.00 | 20.00 |
66 | chr5:64289838 | 0610040J01Rik | T−>A | L−>Q | 0.00 | 24.00 |
67 | chr5:90672580 | Ankrd17 | A−>G | *−>Q | 0.00 | 28.89 |
68 | chr5:109231028 | Vmn2r8 | T−>C | E−>G | 0.00 | 20.83 |
69 | chr5:122789758 | Rad9b | A−>G | L−>S | 0.00 | 37.93 |
70 | chr5:138473740 | Smok3a | A−>G | Q−>R | 0.00 | 32.00 |
71 | chr5:142948192 | C330006K01Rik | G−>A | G−>R | 0.00 | 22.86 |
72 | chr5:146996767 | 1700001J03Rik | C−>T | R−>H | 0.00 | 30.56 |
73 | chr6:67242225 | Il12rb2 | T−>C | Y−>C | 0.00 | 21.21 |
74 | chr6:67423944 | Il23r | T−>C | T−>A | 0.00 | 26.09 |
75 | chr6:72529697 | Elmod3 | T−>C | H−>R | 0.00 | 25.00 |
76 | chr6:123355291 | Vmn2r20 | G−>A | A−>V | 0.00 | 24.00 |
77 | chr6:124820464 | Cd4 | G−>A | P−>S | 0.00 | 30.77 |
78 | chr6:128334974 | 4933413G19Rik | G−>A | G−>R | 0.00 | 23.53 |
79 | chr6:129369539 | Clec9a | A−>G | N−>S | 0.00 | 40.91 |
80 | chr6:132907129 | Tas2r131 | C−>T | R−>Q | 0.00 | 26.09 |
81 | chr6:141942744 | Gm6614 | C−>T | D−>N | 0.00 | 32.14 |
82 | chr6:142186044 | Slco1a5 | G−>A | S−>L | 0.00 | 25.00 |
83 | chr6:142186083 | Slco1a5 | G−>T | P−>H | 0.00 | 27.27 |
84 | chr6:142201619 | Slco1a5 | T−>G | D−>A | 0.00 | 35.29 |
85 | chr6:142251831 | Iapp | G−>C | S−>T | 0.00 | 26.92 |
86 | chr7:3794286 | Pira2 | A−>G | S−>P | 0.00 | 26.09 |
87 | chr7:7278011 | Vmn2r30 | T−>A | N−>I | 0.00 | 22.22 |
88 | chr7:10859910 | Vmn1r66 | G−>A | H−>Y | 0.00 | 38.10 |
89 | chr7:11333654 | Vmn1r71 | G−>A | T−>I | 0.00 | 50.00 |
90 | chr7:12738597 | Vmn1r78 | T−>C | F−>S | 0.00 | 20.00 |
91 | chr7:17743709 | Ceacam3 | C−>G | L−>V | 0.00 | 20.00 |
92 | chr7:17743712 | Ceacam3 | A−>C | I−>L | 0.00 | 22.50 |
93 | chr7:18337478 | Ceacam5 | G−>A | R−>Q | 0.00 | 22.73 |
94 | chr7:18662759 | Ceacam12 | G−>C | G−>A | 0.00 | 26.09 |
95 | chr7:19672969 | Dmpk | G−>A | A−>T | 0.00 | 33.33 |
96 | chr7:26134047 | Megf8 | C−>T | H−>Y | 0.00 | 20.45 |
97 | chr7:26261696 | Ceacam1 | C−>G | A−>P | 0.00 | 20.00 |
98 | chr7:29779122 | Map4k1 | T−>C | C−>R | 0.00 | 21.05 |
99 | chr7:31370138 | Wbp7 | C−>A | A−>S | 0.00 | 31.82 |
100 | chr7:31374957 | Zbtb32 | C−>T | A−>T | 0.00 | 30.77 |
101 | chr7:31391976 | Upk1a | G−>A | T−>I | 0.00 | 31.03 |
102 | chr7:31696435 | Mag | C−>T | V−>I | 0.00 | 40.00 |
103 | chr7:48299575 | Gm4884 | G−>T | A−>S | 0.00 | 21.28 |
104 | chr7:48299666 | Gm4884 | A−>C | H−>P | 0.00 | 22.22 |
105 | chr7:51608348 | Shank1 | G−>A | G−>S | 0.00 | 20.00 |
106 | chr7:54720024 | Mrgpra2b | T−>A | H−>L | 0.00 | 42.42 |
107 | chr7:55424237 | Mrgprb5 | A−>G | I−>T | 0.00 | 30.43 |
108 | chr7:55424238 | Mrgprb5 | T−>A | I−>F | 0.00 | 29.17 |
109 | chr7:86855301 | Kif7 | C−>T | R−>H | 0.00 | 42.42 |
110 | chr7:89455314 | Sh3gl3 | T−>G | S−>A | 0.00 | 40.00 |
111 | chr7:108978612 | Inppl1 | G−>A | H−>Y | 0.00 | 35.29 |
112 | chr7:109584206 | Stim1 | T−>A | L−>H | 0.00 | 24.14 |
113 | chr7:109762976 | Olfr553 | G−>T | L−>M | 0.00 | 23.26 |
114 | chr7:109832588 | Trim68 | T−>C | I−>V | 0.00 | 32.00 |
115 | chr7:109862636 | Olfr33 | A−>G | F−>S | 0.00 | 27.78 |
116 | chr7:109872941 | Olfr559 | A−>T | I−>N | 0.00 | 23.53 |
117 | chr7:110121916 | Olfr577 | C−>T | A−>T | 0.00 | 34.48 |
118 | chr7:110234973 | Olfr584 | T−>C | F−>L | 0.00 | 22.58 |
119 | chr7:110336027 | Olfr592 | A−>G | H−>R | 0.00 | 33.33 |
120 | chr7:110399181 | Dub2a | C−>G | E−>Q | 0.00 | 42.31 |
121 | chr7:110566860 | Usp17l5 | C−>A | P−>T | 0.00 | 25.00 |
122 | chr7:111161069 | Olfr639 | A−>G | I−>T | 0.00 | 25.71 |
123 | chr7:111161070 | Olfr639 | T−>C | I−>V | 0.00 | 27.78 |
124 | chr7:111297423 | Ubqlnl | C−>G | Q−>H | 0.00 | 23.33 |
125 | chr7:111298754 | Ubqlnl | T−>G | T−>P | 0.00 | 31.58 |
126 | chr7:111302579 | E030002O03Rik | A−>G | V−>A | 0.00 | 20.00 |
127 | chr7:111560797 | Trim30a | G−>C | T−>S | 0.00 | 33.33 |
128 | chr7:111793632 | Olfr658 | T−>C | T−>A | 0.00 | 25.00 |
129 | chr7:112009277 | Dub1 | C−>T | R−>C | 0.00 | 35.00 |
130 | chr7:112041695 | Olfr666 | G−>A | A−>V | 0.00 | 29.03 |
131 | chr7:112123974 | Olfr671 | C−>A | S−>I | 0.00 | 20.00 |
132 | chr7:112462829 | Olfr689 | G−>T | A−>S | 0.00 | 21.95 |
133 | chr7:112708033 | Apbb1 | G−>A | S−>F | 0.00 | 26.32 |
134 | chr7:114029870 | Olfr706 | G−>A | L−>F | 0.00 | 23.08 |
135 | chr7:114218082 | Olfr714 | G−>A | V−>I | 0.00 | 21.43 |
136 | chr7:115302565 | Olfr485 | C−>T | G−>E | 0.00 | 28.00 |
137 | chr7:115399125 | Olfr488 | T−>C | K−>E | 0.00 | 25.00 |
138 | chr7:115968770 | Olfr514 | T−>C | T−>A | 0.00 | 25.00 |
139 | chr7:116859980 | BC051019 | G−>A | T−>I | 0.00 | 26.09 |
140 | chr7:135022359 | Zfp646 | C−>G | L−>V | 0.00 | 23.68 |
141 | chr7:135024297 | Zfp646 | G−>A | E−>K | 0.00 | 20.83 |
142 | chr7:135026037 | Zfp646 | A−>G | S−>G | 0.00 | 28.89 |
143 | chr8:4213992 | BC068157 | G−>A | P−>L | 0.00 | 52.38 |
144 | chr8:80770162 | Ttc29 | C−>T | P−>L | 0.00 | 22.86 |
145 | chr8:86691455 | 4930432K21Rik | C−>A | P−>T | 0.00 | 37.50 |
146 | chr8:112256157 | Atxn1l | C−>G | V−>L | 0.00 | 21.88 |
147 | chr9:21085574 | Kri1 | T−>C | K−>E | 0.00 | 35.71 |
148 | chr9:21733911 | Ccdc159 | G−>T | S−>I | 0.00 | 51.72 |
149 | chr9:22004013 | Zfp872 | C−>T | L−>F | 0.00 | 34.88 |
150 | chr9:22005064 | Zfp872 | G−>A | G−>E | 0.00 | 34.15 |
151 | chr9:22005066 | Zfp872 | T−>C | *−>R | 0.00 | 32.50 |
152 | chr9:22058381 | Zfp599 | C−>T | M−>I | 0.00 | 34.62 |
153 | chr9:35646988 | 9230110F15Rik | A−>G | V−>A | 0.00 | 22.22 |
154 | chr9:36671150 | Fez1 | A−>C | E−>D | 0.00 | 34.62 |
155 | chr9:37869528 | Olfr885 | G−>A | V−>M | 0.00 | 27.27 |
156 | chr9:41932183 | Sorl1 | C−>T | S−>N | 0.00 | 32.35 |
157 | chr9:44073278 | Nlrx1 | A−>C | F−>V | 0.00 | 25.00 |
158 | chr9:45557815 | Dscaml1 | G−>C | K−>N | 0.00 | 42.86 |
159 | chr9:50490277 | Dixdc1 | C−>T | R−>Q | 0.00 | 21.74 |
160 | chr9:55821819 | Rfpl3s | G−>A | T−>M | 0.00 | 29.03 |
161 | chr9:58347114 | 6030419C18Rik | G−>A | A−>T | 0.00 | 24.14 |
162 | chr9:120873710 | Ulk4 | T−>C | I−>V | 0.00 | 23.68 |
163 | chr10:18244674 | Nhsl1 | G−>C | C−>S | 0.00 | 21.88 |
164 | chr10:18722769 | Tnfaip3 | A−>G | L−>P | 0.00 | 28.00 |
165 | chr10:51201543 | Gp49a | C−>T | P−>S | 0.00 | 22.22 |
166 | chr10:51201551 | Gp49a | T−>A | H−>Q | 0.00 | 20.00 |
167 | chr10:51203657 | Gp49a | T−>C | Y−>H | 0.00 | 27.50 |
168 | chr10:51203677 | Gp49a | T−>G | N−>K | 0.00 | 44.74 |
169 | chr10:53257912 | Mcm9 | A−>T | S−>T | 0.00 | 37.50 |
170 | chr10:61892173 | Supv3l1 | C−>T | D−>N | 0.00 | 31.43 |
171 | chr10:62301871 | Tet1 | T−>C | E−>G | 0.00 | 27.59 |
172 | chr10:62534718 | Pbld1 | G−>T | G−>V | 0.00 | 26.67 |
173 | chr10:62534721 | Pbld1 | G−>A | G−>E | 0.00 | 26.67 |
174 | chr10:69997479 | Fam13c | T−>G | S−>A | 0.00 | 23.68 |
175 | chr10:82654374 | Chst11 | G−>A | G−>S | 0.00 | 23.08 |
176 | chr10:85391311 | Ascl4 | G−>C | G−>R | 0.00 | 28.57 |
177 | chr10:99909744 | Tmtc3 | C−>T | R−>K | 0.00 | 30.00 |
178 | chr10:99914062 | Tmtc3 | C−>T | R−>K | 0.00 | 44.83 |
179 | chr10:100031465 | Cep290 | A−>C | M−>L | 0.00 | 47.62 |
180 | chr10:128448679 | Olfr763 | T−>A | C−>S | 0.00 | 20.00 |
181 | chr11:5587351 | Ankrd36 | G−>A | V−>I | 0.00 | 33.33 |
182 | chr11:5587391 | Ankrd36 | A−>T | K−>I | 0.00 | 22.22 |
183 | chr11:6501551 | Nacad | G−>A | P−>S | 0.00 | 39.39 |
184 | chr11:23264045 | Usp34 | A−>T | E−>D | 0.00 | 28.57 |
185 | chr11:29429943 | Mtif2 | A−>G | Q−>R | 0.00 | 25.93 |
186 | chr11:29607190 | Rtn4 | G−>C | S−>T | 0.00 | 25.00 |
187 | chr11:29607841 | Rtn4 | C−>T | A−>V | 0.00 | 31.25 |
188 | chr11:29646793 | Eml6 | G−>C | L−>V | 0.00 | 37.14 |
189 | chr11:32184064 | Hba-a1 | G−>C | G−>A | 0.00 | 28.00 |
190 | chr11:35622812 | Rars | G−>T | A−>E | 0.00 | 22.22 |
191 | chr11:48988354 | Btnl9 | T−>C | Q−>R | 0.00 | 39.13 |
192 | chr11:52216575 | 9530068E07Rik | C−>T | A−>V | 0.00 | 34.38 |
193 | chr11:62078872 | Adora2b | G−>A | R−>H | 0.00 | 25.71 |
194 | chr11:67688822 | Usp43 | T−>C | M−>V | 0.00 | 24.00 |
195 | chr11:69010998 | Alox8 | A−>G | V−>A | 0.00 | 21.43 |
196 | chr11:70584746 | Zfp3 | C−>A | P−>T | 0.00 | 20.83 |
197 | chr11:70995613 | Nlrp1b | A−>T | F−>Y | 0.00 | 21.74 |
198 | chr11:70995614 | Nlrp1b | A−>G | F−>L | 0.00 | 23.81 |
199 | chr11:70995616 | Nlrp1b | A−>C | I−>R | 0.00 | 22.73 |
200 | chr11:72984698 | P2rx5 | C−>T | A−>V | 0.00 | 25.00 |
201 | chr11:96214596 | Hoxb2 | G−>A | E−>K | 0.00 | 61.90 |
202 | chr11:96772447 | Cdk5rap3 | C−>T | V−>I | 0.00 | 47.50 |
203 | chr11:101045277 | Cntnap1 | C−>T | S−>L | 0.00 | 31.25 |
204 | chr11:102935952 | Plcd3 | A−>T | D−>E | 0.00 | 23.81 |
205 | chr11:106174196 | Cd79b | T−>C | M−>V | 0.00 | 22.22 |
206 | chr11:120146302 | Bahcc1 | C−>G | T−>S | 0.00 | 21.43 |
207 | chr12:18521595 | 5730507C01Rik | A−>T | N−>Y | 0.00 | 20.00 |
208 | chr12:21271015 | Asap2 | C−>T | T−>I | 0.00 | 21.43 |
209 | chr12:21379212 | Adam17 | A−>C | S−>A | 0.00 | 20.69 |
210 | chr12:25723341 | Kidins220 | G−>A | G−>S | 0.00 | 31.82 |
211 | chr12:32005994 | Lamb1 | T−>C | V−>A | 0.00 | 25.00 |
212 | chr12:65573550 | Fscb | A−>G | S−>P | 0.00 | 25.00 |
213 | chr12:77031626 | Syne2 | G−>A | R−>K | 0.00 | 20.00 |
214 | chr12:77088037 | Syne2 | A−>G | H−>R | 0.00 | 37.93 |
215 | chr12:77701313 | Spnb1 | G−>C | D−>E | 0.00 | 27.08 |
216 | chr12:77713010 | Spnb1 | A−>T | M−>K | 0.00 | 26.47 |
217 | chr12:80369378 | Zfyve26 | T−>C | Q−>R | 0.00 | 28.00 |
218 | chr12:85333734 | Acot2 | A−>G | T−>A | 0.00 | 22.86 |
219 | chr12:88947186 | Oog1 | G−>A | E−>K | 0.00 | 20.45 |
220 | chr12:111906810 | 1700001K19Rik | T−>G | Q−>P | 0.00 | 29.82 |
221 | chr13:6564252 | Pitrm1 | C−>A | T−>K | 0.00 | 32.14 |
222 | chr13:6604968 | Pfkp | C−>T | V−>M | 0.00 | 34.48 |
223 | chr13:8886000 | Idi1 | T−>C | S−>P | 0.00 | 22.58 |
224 | chr13:8958551 | Idi2 | A−>G | E−>G | 0.00 | 27.27 |
225 | chr13:9150373 | Larp4b | T−>C | L−>S | 0.00 | 34.48 |
226 | chr13:9688439 | Zmynd11 | C−>T | S−>N | 0.00 | 33.33 |
227 | chr13:14097474 | Tbce | G−>A | A−>V | 0.00 | 22.73 |
228 | chr13:23126330 | Vmn1r214 | G−>A | E−>K | 0.00 | 42.86 |
229 | chr13:23126981 | Vmn1r214 | C−>A | Q−>K | 0.00 | 40.91 |
230 | chr13:23309404 | Vmn1r221 | C−>A | L−>I | 0.00 | 30.77 |
231 | chr13:23309956 | Vmn1r221 | C−>G | L−>V | 0.00 | 25.93 |
232 | chr13:23579753 | Btn2a2 | T−>A | I−>L | 0.00 | 34.62 |
233 | chr13:23647236 | Hist1h1d | C−>T | T−>I | 0.00 | 37.50 |
234 | chr13:23855668 | Hist1h1a | C−>T | A−>V | 0.00 | 29.63 |
235 | chr13:25085054 | Mrs2 | T−>C | T−>A | 0.00 | 21.74 |
236 | chr13:40238189 | Ofcc1 | G−>A | P−>S | 0.00 | 20.83 |
237 | chr13:58445640 | Kif27 | T−>A | I−>L | 0.00 | 23.81 |
238 | chr13:70874611 | Adamts16 | C−>T | G−>S | 0.00 | 24.14 |
239 | chr13:70877487 | Adamts16 | G−>C | Q−>E | 0.00 | 20.59 |
240 | chr13:81583863 | Gpr98 | C−>T | E−>K | 0.00 | 34.48 |
241 | chr13:96284081 | F2rl1 | C−>T | V−>I | 0.00 | 25.00 |
242 | chr13:98737291 | Rgnef | G−>C | A−>G | 0.00 | 33.33 |
243 | chr14:45342600 | Gm8267 | A−>T | M−>K | 0.00 | 28.00 |
244 | chr14:50393514 | 3632451O06Rik | A−>G | V−>A | 0.00 | 24.44 |
245 | chr14:51135131 | Olfr742 | A−>G | N−>S | 0.00 | 25.81 |
246 | chr14:55283622 | Acin1 | T−>C | K−>E | 0.00 | 43.18 |
247 | chr14:70175997 | Tnfrsf10b | C−>G | P−>A | 0.00 | 27.78 |
248 | chr14:70176001 | Tnfrsf10b | T−>C | V−>A | 0.00 | 25.00 |
249 | chr14:78484964 | AU021034 | A−>G | C−>R | 0.00 | 22.22 |
250 | chr15:41697429 | Abra | G−>C | L−>V | 0.00 | 24.00 |
251 | chr15:41701040 | Abra | G−>C | L−>V | 0.00 | 27.27 |
252 | chr15:54965030 | Deptor | A−>T | M−>L | 0.00 | 25.00 |
253 | chr15:66523859 | Tg | G−>A | V−>I | 0.00 | 20.00 |
254 | chr15:75937421 | Eppk1 | C−>T | V−>I | 0.00 | 25.00 |
255 | chr15:76539950 | Recql4 | A−>G | L−>P | 0.00 | 20.00 |
256 | chr15:95455328 | Dbx2 | C−>T | V−>M | 0.00 | 25.00 |
257 | chr16:32756226 | Muc4 | C−>G | Q−>E | 0.00 | 24.32 |
258 | chr16:32779211 | Muc4 | C−>A | Q−>K | 0.00 | 23.33 |
259 | chr16:45577986 | Slc9a10 | C−>T | A−>V | 0.00 | 20.00 |
260 | chr16:56668453 | Abi3bp | C−>A | P−>Q | 0.00 | 22.22 |
261 | chr16:58872574 | Olfr176 | C−>G | S−>T | 0.00 | 20.59 |
262 | chr17:6009735 | Synj2 | T−>A | F−>L | 0.00 | 25.81 |
263 | chr17:6037828 | Synj2 | C−>G | H−>D | 0.00 | 25.00 |
264 | chr17:7530924 | Tcp10a | C−>T | P−>S | 0.00 | 29.03 |
265 | chr17:24111200 | Prss30 | A−>C | D−>E | 0.00 | 21.05 |
266 | chr17:24583771 | E4f1 | C−>T | S−>N | 0.00 | 27.27 |
267 | chr17:28021909 | Uhrf1bp1 | G−>A | G−>D | 0.00 | 22.50 |
268 | chr17:31365125 | Ubash3a | C−>T | P−>S | 0.00 | 25.81 |
269 | chr17:31392140 | Rsph1 | G−>T | P−>Q | 0.00 | 25.71 |
270 | chr17:31398701 | Rsph1 | G−>A | T−>M | 0.00 | 30.30 |
271 | chr17:31754132 | Cbs | T−>C | D−>G | 0.00 | 29.03 |
272 | chr17:32758655 | Gm9705 | G−>A | V−>M | 0.00 | 20.00 |
273 | chr17:33158753 | Zfp763 | C−>T | A−>T | 0.00 | 22.86 |
274 | chr17:33472542 | Zfp81 | A−>G | M−>T | 0.00 | 27.59 |
275 | chr17:34087468 | B3galt4 | T−>C | N−>S | 0.00 | 28.21 |
276 | chr17:34338203 | Psmb8 | G−>T | A−>S | 0.00 | 28.57 |
277 | chr17:34870392 | C4b | C−>T | R−>Q | 0.00 | 22.58 |
278 | chr17:34974426 | Dom3z | T−>C | L−>S | 0.00 | 50.00 |
279 | chr17:35267082 | Apom | G−>T | Q−>K | 0.00 | 43.33 |
280 | chr17:35457904 | H2-Q1 | C−>T | P−>L | 0.00 | 32.50 |
281 | chr17:36168621 | H2-T23 | C−>G | R−>T | 0.00 | 28.57 |
282 | chr17:36218438 | H2-Bl | T−>C | H−>R | 0.00 | 27.03 |
283 | chr17:36254622 | H2-T10 | G−>A | P−>S | 0.00 | 33.33 |
284 | chr17:36323554 | H2-T3 | T−>G | M−>L | 0.00 | 21.67 |
285 | chr17:43615815 | Mep1a | T−>C | T−>A | 0.00 | 25.00 |
286 | chr17:43615911 | Mep1a | T−>C | T−>A | 0.00 | 30.00 |
287 | chr17:43822205 | Cyp39a1 | G−>A | G−>R | 0.00 | 28.57 |
288 | chr17:46161537 | Vegfa | G−>A | P−>L | 0.00 | 37.04 |
289 | chr17:46550212 | Zfp318 | A−>G | E−>G | 0.00 | 29.03 |
290 | chr17:46635998 | BC048355 | A−>C | K−>N | 0.00 | 31.82 |
291 | chr17:46893214 | Ptcra | G−>T | R−>S | 0.00 | 37.14 |
292 | chr17:72047254 | Fam179a | T−>G | F−>C | 0.00 | 37.50 |
293 | chr17:80734673 | Arhgef33 | G−>A | A−>T | 0.00 | 26.67 |
294 | chr17:88958139 | Klraq1 | T−>C | M−>T | 0.00 | 29.03 |
295 | chr17:89110812 | Gtf2a1l | G−>A | R−>Q | 0.00 | 31.03 |
296 | chr17:89153211 | Lhcgr | T−>C | T−>A | 0.00 | 57.58 |
297 | chr18:37907724 | Pcdhga10 | C−>A | H−>N | 0.00 | 26.09 |
298 | chr18:38132920 | Arap3 | G−>A | A−>V | 0.00 | 44.83 |
299 | chr18:60977711 | Tcof1 | C−>A | A−>S | 0.00 | 56.00 |
300 | chr18:60992401 | Tcof1 | C−>A | A−>S | 0.00 | 46.43 |
301 | chr18:65901869 | 5330437I02Rik | T−>C | F−>L | 0.00 | 20.59 |
302 | chr18:80326155 | Adnp2 | A−>G | F−>L | 0.00 | 26.83 |
303 | chr18:80389581 | Rbfa | C−>T | A−>T | 0.00 | 32.35 |
304 | chr19:10751147 | Pga5 | C−>G | V−>L | 0.00 | 45.83 |
305 | chr19:11038598 | Ms4a10 | C−>T | V−>I | 0.00 | 20.69 |
306 | chr19:18912582 | Trpm6 | A−>G | M−>V | 0.00 | 21.88 |
307 | chr19:25696788 | Dmrt3 | C−>T | T−>M | 0.00 | 29.63 |
308 | chr7:111207208 | Olfr643 | G−>A | R−>* | 0.00 | 29.41 |
Similar gene expression profile in lv-iPSCs with or without ionizing radiation
Gene expression levels in cells exposed or not to ionizing radiation (IR) for the indicated periods. a Heatmap showing Pearson’s correlation coefficients relating expression levels between irradiated and non-irradiated cells. b Volcano plots of genes expressed in irradiated and non-irradiated cells, showing genes significantly up-regulated (red dots) or down-regulated (green dots) in irradiated cells. Differentially expressed genes were filtered based on FDR < 0.05. c, d Histograms of gene ontology classifications of differentially expressed genes in irradiated (c) ESCs and (d) iPSCs. e Heat maps showing the expression level of DNA damage repair-associated genes in irradiated (+) and non-irradiated (−) cells. Blue indicates lowest expression; fuchsia, highest. BER base excision repair, HR, homologous recombination, MMR mismatch repair, NER nucleotide excision repair, NHEJ non-homologous end joining
Expression levels of genes involved in DNA damage repair pathways were higher in lv-iPSCs and ESCs than in MEFs, and ionizing radiation did not substantially alter the expression of these genes (Fig. 3e). Thus the genomic instability of lv-iPSCs is unlikely to reflect changes in the expression level of genes involved in DNA damage repair.
Weaker DNA damage repair response to ionizing radiation in lv-iPSCs
The phosphorylation level of DNA repair-associated proteins. a Quantification of the numbers of γ-H2AX foci in lv-iPSCs, ESCs and MEFs. Error bars represent the standard error of the mean (SEM) for the numbers of γ-H2AX foci per nucleus based on 4–5 fields, each containing approximately 20–30 cells. Significance of differences was assessed using Student’s t test. **P < 0.01 (three independent experiments). b Western blot analysis of phosphorylated ATM (p-ATM) and phosphorylated catalytic subunit of DNA protein kinase (p-DNA-PKcs) in lv-iPSCs and ESCs before and after ionizing irradiation. c Western blot analysis of the trimethylation level of H3K9 in lv-iPSCs and ESCs before and after ionizing irradiation
Next we tested whether the lower genomic stability of lv-iPSCs reflects deficiency in the error-free HR repair pathway. Indeed, we found ATM phosphorylation to be defective in lv-iPSCs (Fig. 4b) [30, 41]. We also found lower levels of H3K9me3, which recruits repair proteins to double-strand breaks, in irradiated lv-iPSCs than in irradiated ESCs or MEFs (Fig. 4c). All together, these findings may help explain the higher mutation rate of lv-iPSCs.
Lower genomic stability in lv-iPSCs than ci-iPSCs
High genome stability of ci-iPSCs. a Western blot analysis of phosphorylated ATM (p-ATM) in ci-iPS, lv-iPS and ESCs before and after ionizing irradiation. b Circos plot showing genetic alterations in irradiated ci-iPSCs and ESCs, based on the corresponding untreated cells as a reference. Chromosome numbers are indicated as the outermost labels. c Histograms showing the number of SNVs in each genomic region of irradiated lv-iPSCs, ci-iPSCs and MEFs. CDS coding sequence, SNV single-nucleotide variants, UTR untranslated region. d Histograms showing the numbers of SNVs in the coding regions of irradiated lv-iPSCs, ci-iPSCs and MEFs
lv-iPSCs can tolerate more genomic DNA variation
Greater tolerance of genomic DNA variation in lv-iPSCs. a Flow cytometric analysis of apoptosis rate in lv-iPSCs, ESCs and MEFs following ionizing irradiation (IR) for the indicated periods. 7-AAD 7-amino-actinomycin. b Cell proliferation rate (based on BrdU incorporation) in lv-iPSCs, ESCs and MEFs exposed to ionizing irradiation for the indicated periods. Each point represents a mean of three replicates **P < 0.01. c Analysis of cell cycle distribution in lv-iPS, ESCs and MEFs exposed to ionizing radiation for the indicated periods
lv-iPSCs are more susceptible to DNA damage
Genome stability of mice derived from lv-iPSCs or ESCs following exposure to ionizing radiation (IR). Controls were C57 mice. a Mice were generated from lv-iPSCs or ESCs through tetraploid embryo complementation. Representative results from three independent experiments are shown. b Examples of bones from the three types of mice, from which marrow cells were extracted. c Box plots showing the percentage of impaired bone marrow cells in each mouse strain. DNA damage was evaluated using single-cell gel electrophoresis **P < 0.01. d Box plots showing the percentage of Tail DNA in impaired cells as a measure of DNA damage. Tail DNA% = Tail DNA intensity/Cell DNA Intensity × 100%. CASP software was used to calculate tail moment based on 50–100 randomly selected cells per sample
Diagram illustrating factors influencing the genome stability of iPSCs. Environmental factors contribute to genomic variations in lv-iPSCs. In response to double-strand DNA breaks, lv-iPSCs always adopt the error-prone NHEJ repair pathway. The resulting low fidelity of DNA repair makes the lv-iPSC genome unstable and the cells more vulnerable to environmental stress. Genomic stability of iPSCs appears to depend on the method used to generate them: ci-iPSCs show greater stability than lv-iPSCs. HR homologous recombination repair pathway, IR ionizing radiation, NHEJ non-homologous end joining
Discussion
Reprogramming to generate iPSCs more efficiently [29, 42–51] has been linked to the accumulation of genomic abnormalities [52–59]. This poses a problem for the use of iPSCs, since mice derived from such cells can tolerate the accumulation of somatic mutations for up to six generations [60]. In the present study, we used whole-genome sequencing to compare the genomic stability of iPSCs prepared using lentivirus or chemically, and to benchmark that stability against ESCs and MEFs. We found that ionizing irradiation led to the highest rate of somatic mutations and short indels in lv-iPSCs, and this correlated with low levels of ATM phosphorylation, indicating low fidelity of DNA damage repair [41]. Experiments in vitro and in mice derived from lv-iPSCs showed that this type of pluripotent cell tolerates genomic mutations better than the other cell types evaluated.
Although iPSCs resemble ESCs in morphology, gene expression profile and in vitro differentiation capacity, they differ substantially in genomic stability. The low fidelity of DNA repair observed in our study suggests that irradiation of lv-iPSCs induces a high rate of genomic abnormalities, which is less likely to trigger apoptosis in these cells and is therefore more likely to be tolerated, thus leading to a high rate of tumorigenesis in vivo. Compromised error-free HR pathway of DNA damage repair in lv-iPSCs may help explain the relatively high genomic instability in these cells. Indeed, inhibiting the HR pathway in iPSCs has been shown to destabilize the genome [61].
Our results suggest that the epigenetic status of iPSCs may contribute to, or modulate, their genomic instability. Variation in levels of H3K9me3 and phosphorylated ATM among iPSCs may mean that cells vary in their reliance on DNA damage repair pathways, which vary in their fidelity. Future studies should further examine the potential involvement of epigenetics and other factors in iPSC genomic instability.
Future work is also needed to clarify to what extent factors that are intrinsic or extrinsic to stem cells determine the risk of malignant transformation. Tomasetti et al. found that cancer risk in certain tissues correlated strongly with the number of divisions that the stem cells had undergone, suggesting that the accumulation of genomic mutations is primarily responsible for high risk of tumorigenesis [62]. Another study, in contrast, suggested that intrinsic factors account for only 10%–30% of cancer risk, with the majority of the risk due to extrinsic factors [63]. The results from the present study suggest that extrinsic factors induce more genomic mutations than intrinsic factors in lv-iPSCs. The high rate of tumorigenesis of iPSCs in vivo suggests that extrinsic factors strongly contribute to cancer risk and carcinogenesis.
Conclusions
The present study demonstrated a low level of DNA damage repair in iPSCs. Ionizing radiation induced more somatic mutations and short indels in iPSCs than in ESCs or MEFs. Genome stability was higher in iPSCs induced chemically than in iPSCs induced with lentivirus. The high genome instability of lv-iPSCs appears to reflect increased NHEJ and decreased HR pathways of DNA damage repair, and could contribute to the high rate of tumorigenesis in vivo.
Notes
Declarations
Authors’ contributions
YS, QZ and JC conceived this study. MZ and LW contributed to its design. MZ, GL, KA, CY, HL, FD, XH and YL performed experiments, while MZ and GL performed bioinformatic analyses. JC assisted with bioinformatic analysis and interpretation. YS and MZ interpreted the data and wrote the paper with the assistance of the other authors. QZ, DP, and XX provided critical technical assistance and expertise. All authors read and approved the final manuscript.
Acknowledgements
We thank Professor Qi Zhou from the Institute of Zoology of the Chinese Academy of Sciences for generously supplying iPS- and ES-derived mice. We thank Professor Duanqing Pei from the Guangzhou Institutes of Biomedicine and Health of the Chinese Academy of Sciences for providing the lv-iPSC line, and Xin Xie from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences for providing the ci-iPSC line. We are also grateful to our laboratory colleagues for their assistance with experiments and manuscript preparation.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The raw sequencing data reported in this manuscript are publicly available at the Genome Sequence Archive (http://gsa.big.ac.cn) under Accession Number CRA000695.
All data generated and analyzed are included in the article to support the conclusions.
Consent for publication
Not applicable.
Ethics approval and consent to participate
This study was approved by the Ethics Committee of the Beijing Institute of Genomics and the School of Life Sciences at the Chinese Academy of Sciences.
Funding
This work was supported by the Precision Medicine Research Program of the Chinese Academy of Sciences (KJZD-EW-L14), Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01040407), National Natural Science Foundation of China (31471395, 91019024, 31540033 and 31100558), National Basic Research Program of China (973 Program, 2012CB518302 and 2013CB911001) and 100 Talents Project.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
References
- Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282(5391):1145–7.PubMedView ArticleGoogle Scholar
- Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.PubMedView ArticleGoogle Scholar
- de Lazaro I, Yilmazer A, Kostarelos K. Induced pluripotent stem (iPS) cells: a new source for cell-based therapeutics? J Control Release. 2014;185:37–44.PubMedView ArticleGoogle Scholar
- Amabile G, Meissner A. Induced pluripotent stem cells: current progress and potential for regenerative medicine. Trends Mol Med. 2009;15(2):59–68.PubMedView ArticleGoogle Scholar
- Yamanaka S. A fresh look at iPS cells. Cell. 2009;137(1):13–7.PubMedView ArticleGoogle Scholar
- Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer. 2008;8(12):976–90.PubMedView ArticleGoogle Scholar
- Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008;40(5):499–507.PubMedPubMed CentralView ArticleGoogle Scholar
- Malchenko S, Galat V, Seftor EA, Vanin EF, Costa FF, Seftor RE, et al. Cancer hallmarks in induced pluripotent cells: new insights. J Cell Physiol. 2010;225(2):390–3.PubMedPubMed CentralView ArticleGoogle Scholar
- Rowland BD, Peeper DS. KLF4, p21 and context-dependent opposing forces in cancer. Nat Rev Cancer. 2006;6(1):11–23.PubMedView ArticleGoogle Scholar
- Ghosh Z, Huang M, Hu S, Wilson KD, Dey D, Wu JC. Dissecting the oncogenic and tumorigenic potential of differentiated human induced pluripotent stem cells and human embryonic stem cells. Cancer Res. 2011;71(14):5030–9.PubMedPubMed CentralView ArticleGoogle Scholar
- Blasco MA, Serrano M, Fernandez-Capetillo O. Genomic instability in iPS: time for a break. EMBO J. 2011;30(6):991–3.PubMedPubMed CentralView ArticleGoogle Scholar
- Pasi CE, Dereli-Oz A, Negrini S, Friedli M, Fragola G, Lombardo A, et al. Genomic instability in induced stem cells. Cell Death Differ. 2011;18(5):745–53.PubMedPubMed CentralView ArticleGoogle Scholar
- Pera MF. Stem cells: the dark side of induced pluripotency. Nature. 2011;471(7336):46–7.PubMedView ArticleGoogle Scholar
- Ronen D, Benvenisty N. Genomic stability in reprogramming. Curr Opin Genet Dev. 2012;22(5):444–9.PubMedView ArticleGoogle Scholar
- Sarig R, Rotter V. Can an iPS cell secure its genomic fidelity? Cell Death Differ. 2011;18(5):743–4.PubMedPubMed CentralView ArticleGoogle Scholar
- von Joest M, Bua Aguin S, Li H. Genomic stability during cellular reprogramming: mission impossible? Mutat Res. 2016;788:12–6.View ArticleGoogle Scholar
- Friedberg EC. A history of the DNA repair and mutagenesis field The discovery of base excision repair. DNA Repair. 2016;37:A35–9.PubMedView ArticleGoogle Scholar
- Lieber MR. The mechanism of human nonhomologous DNA end joining. J Biol Chem. 2008;283(1):1–5.PubMedView ArticleGoogle Scholar
- Filippo JS, Sung P, Klein H. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem. 2008;77:229–57.View ArticleGoogle Scholar
- Weeden CE, Chen YS, Ma SB, Hu YF, Ramm G, Sutherland KD, et al. Lung basal stem cells rapidly repair DNA damage using the error-prone nonhomologous end-joining pathway. PLoS Biol. 2017;15(1):e2000731.PubMedPubMed CentralView ArticleGoogle Scholar
- Long Y, Wang M, Gu HF, Xie X. Bromodeoxyuridine promotes full-chemical induction of mouse pluripotent stem cells. Cell Res. 2015;25(10):1171–4.PubMedPubMed CentralView ArticleGoogle Scholar
- Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis. 2002;23(5):687–96.PubMedView ArticleGoogle Scholar
- Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet. 2001;27(3):247–54.PubMedView ArticleGoogle Scholar
- Lombard DB, Chua KF, Mostoslavsky R, Franco S, Gostissa M, Alt FW. DNA repair, genome stability, and aging. Cell. 2005;120(4):497–512.PubMedView ArticleGoogle Scholar
- Zha S, Alt FW, Cheng HL, Brush JW, Li G. Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells. Proc Natl Acad Sci USA. 2007;104(11):4518–23.PubMedView ArticleGoogle Scholar
- Rooney S, Alt FW, Lombard D, Whitlow S, Eckersdorff M, Fleming J, et al. Defective DNA repair and increased genomic instability in artemis-deficient murine cells. J Exp Med. 2003;197(5):553–65.PubMedPubMed CentralView ArticleGoogle Scholar
- Esteban MA, Wang T, Qin BM, Yang JY, Qin DJ, Cai JL, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell. 2010;6(1):71–9.PubMedView ArticleGoogle Scholar
- Zhang M, Yang C, Liu H, Sun Y. Induced pluripotent stem cells are sensitive to DNA damage. Genomics Proteomics Bioinf. 2013;11(5):320–6.View ArticleGoogle Scholar
- Huangfu DW, Maehr R, Guo WJ, Eijkelenboom A, Snitow M, Chen AE, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008;26(7):795–7.PubMedView ArticleGoogle Scholar
- Sun Y, Xu Y, Roy K, Price BD. DNA damage-induced acetylation of lysine 3016 of ATM activates ATM kinase activity. Mol Cell Biol. 2007;27(24):8502–9.PubMedPubMed CentralView ArticleGoogle Scholar
- Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754–60.PubMedPubMed CentralView ArticleGoogle Scholar
- Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078–9.PubMedPubMed CentralView ArticleGoogle Scholar
- McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297–303.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36.PubMedPubMed CentralView ArticleGoogle Scholar
- Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9.PubMedPubMed CentralView ArticleGoogle Scholar
- Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7(3):562–78.PubMedPubMed CentralView ArticleGoogle Scholar
- Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004;5(10):R80.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhao XY, Lv Z, Li W, Zeng F, Zhou Q. Production of mice using iPS cells and tetraploid complementation. Nat Protoc. 2010;5(5):963–71.PubMedView ArticleGoogle Scholar
- Shu J, Zhang K, Zhang MJ, Yao AZ, Shao SD, Du FX, et al. GATA family members as inducers for cellular reprogramming to pluripotency. Cell Res. 2015;25(2):169–80.PubMedPubMed CentralView ArticleGoogle Scholar
- Johnson RD, Jasin M. Double-strand-break-induced homologous recombination in mammalian cells. Biochem Soc Trans. 2001;29(Pt 2):196–201.PubMedView ArticleGoogle Scholar
- Lavin MF, Birrell G, Chen P, Kozlov S, Scott S, Gueven N. ATM signaling and genomic stability in response to DNA damage. Mutat Res. 2005;569(1–2):123–32.PubMedView ArticleGoogle Scholar
- Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–72.PubMedView ArticleGoogle Scholar
- Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced pluripotent stem cells generated without viral integration. Science. 2008;322(5903):945–9.PubMedPubMed CentralView ArticleGoogle Scholar
- Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature. 2009;458(7239):771–5.PubMedPubMed CentralView ArticleGoogle Scholar
- Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hamalainen R, et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009;458(7239):766–70.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhou W, Freed CR. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells. 2009;27(11):2667–74.PubMedView ArticleGoogle Scholar
- Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell. 2009;4(5):381–4.PubMedView ArticleGoogle Scholar
- Warren L, Manos PD, Ahfeldt T, Loh Y-H, Li H, Lau F, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010;7(5):618–30.PubMedPubMed CentralView ArticleGoogle Scholar
- Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell. 2011;8(6):633–8.PubMedView ArticleGoogle Scholar
- Hou PP, Li YQ, Zhang X, Liu C, Guan JY, Li HG, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013;341(6146):651–4.PubMedView ArticleGoogle Scholar
- Shu J, Wu C, Wu Y, Li Z, Shao S, Zhao W, et al. Induction of pluripotency in mouse somatic cells with lineage specifiers. Cell. 2013;153(5):963–75.PubMedPubMed CentralView ArticleGoogle Scholar
- Stadtfeld M, Apostolou E, Akutsu H, Fukuda A, Follett P, Natesan S, et al. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature. 2010;465(7295):175–81.PubMedPubMed CentralView ArticleGoogle Scholar
- Laurent LC, Ulitsky I, Slavin I, Tran H, Schork A, Morey R, et al. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell. 2011;8(1):106–18.PubMedPubMed CentralView ArticleGoogle Scholar
- Mayshar Y, Ben-David U, Lavon N, Biancotti JC, Yakir B, Clark AT, et al. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell. 2010;7(4):521–31.PubMedView ArticleGoogle Scholar
- Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature. 2011;471(7336):68–73.PubMedPubMed CentralView ArticleGoogle Scholar
- Polo JM, Liu S, Figueroa ME, Kulalert W, Eminli S, Tan KY, et al. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol. 2010;28(8):848–55.PubMedPubMed CentralView ArticleGoogle Scholar
- Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, et al. Somatic coding mutations in human induced pluripotent stem cells. Nature. 2011;471(7336):63–7.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, et al. Epigenetic memory in induced pluripotent stem cells. Nature. 2010;467(7313):285–90.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim K, Zhao R, Doi A, Ng K, Unternaehrer J, Cahan P, et al. Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells. Nat Biotechnol. 2012;30(1):1117–9.View ArticleGoogle Scholar
- Gao S, Zheng C, Chang G, Liu W, Kou X, Tan K, et al. Unique features of mutations revealed by sequentially reprogrammed induced pluripotent stem cells. Nat Commun. 2015;6:6318.PubMedView ArticleGoogle Scholar
- Thompson LH, Schild D. The contribution of homologous recombination in preserving genome integrity in mammalian cells. Biochimie. 1999;81(1–2):87–105.PubMedView ArticleGoogle Scholar
- Tomasetti C, Vogelstein B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science. 2015;347(6217):78–81.PubMedPubMed CentralView ArticleGoogle Scholar
- Wu S, Powers S, Zhu W, Hannun YA. Substantial contribution of extrinsic risk factors to cancer development. Nature. 2016;529(7584):43–7.PubMedView ArticleGoogle Scholar
